PROJECT SUMMARY Glioblastoma multiforme (GBM) is the most common and lethal brain malignancy with a median survival of only one year after diagnosis. Our current knowledge of the underlying basis of GBM centers mostly on several recurrent mutations in specific genes. However, non-genetic factors contributing to GBM development and progression are largely unknown. Due to our poor understanding of GBM biology, treatment options are limited to chemotherapy (temozolomide, TMZ) combined with radiotherapy. Thus, new therapeutic approaches are desperately needed to treat this deadly tumor. Emerging evidence has demonstrated that aberrant RNA splicing due to splice site mutations and/or splicing factor mutations drives oncogenic gene expression in multiple types of solid tumors and hematologic malignancies. In GBM, a few RNA splicing factors, including polypyrimidine tract-binding protein 1 (PTBP1) and hnRNP A2B1 have been recently identified as driving factors in oncogenic splicing, indicating that RNA splicing is a critical, yet to be explored, mechanism that governs a broad range of oncogenic gene expression. Our extensive preliminary data demonstrated that SON, a large nuclear speckle protein possessing DNA- and RNA-binding abilities, is highly upregulated in GBM patient samples, and there is a strong correlation between SON upregulation and short patient survival. We found that SON facilitates expression of PTBP1, thereby activating the PTBP1-meditated oncogenic splicing program. In contrast, SON inhibits the expression of PTBP2, a splicing factor required for neural exon inclusion and neural differentiation. We further revealed that SON regulates the intron removal process at the constitutive splice site in the PTBP1 transcript and regulates cassette exon inclusion/skipping at the alternative splice site in the PTBP2 transcript. We also demonstrated that SON knockdown markedly inhibits GBM cell growth and neural stem cell gene expression, and SON depletion renders patient-derived glioma stem cells (GSCs) sensitive to TMZ in vitro. Based on our preliminary data, we hypothesize that SON is a master RNA splicing regulator positioned at the apex of the splicing factor hierarchy that affects both constitutive and alternative RNA splicing, consequently turning on the oncogenic splicing program and blocking neural splicing. Thus, SON could represent a promising novel therapeutic target for GBM. To test this hypothesis, we propose to dissect the molecular mechanisms of SON functions in the regulation of constitutive and alternative RNA splicing in GBM (Aim 1), and to determine the therapeutic potential of targeting SON in vivo (Aim 2). Successful completion of this proposed study will significantly advance our knowledge of abnormal gene expression in GBM and provide a fundamental rationale for future endeavors to develop SON inhibitors.